Near Infrared Fluorescent Contrast Agent And Method For Fluorescence Imaging

ABSTRACT

A near infrared fluorescent contrast agent which is excellent in permeability in a living tissue and enables specific imaging of tumor and/or blood vessel, comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 7 , and R 8  represent a C 1 -C 10  alkyl group or the like; R 3 , R 4 , R 5 , R 6 , R 9 , R 10 , R 11 , and R 12  represent a hydrogen atom, a C 1 -C 6  alkyl group, an aryl group or the like; X 1  and X 2  represent a C 1 -C 15  alkyl group or an aryl group and X 1  and X 2  in total have 0 to 4 carboxyl groups; m 1 , m 2 , and m 3  represents 0 or 1; L 1  to L 7  independently represent a methine group; M represents a hydrogen atom, a metal, or a quaternary ammonium salt; and n represents an integer of 1 to 7 necessary for neutralizing charge.

TECHNICAL FIELD

The present invention relates to a near infrared fluorescent contrastagent, and a method of fluorescence imaging using said near infraredfluorescent contract agent.

BACKGROUND ART

In treating disease, it is important to detect morphological andfunctional changes caused by the disease in the living body at an earlystage of the disease. Especially for treatment of a cancer, to know thesite and size of the tumor beforehand is an extremely important means todetermine strategies and protocols for future treatment. Methods so farapplied include biopsy by puncture and the like, as well as imagingdiagnosis such as X-ray imaging, MRI, ultrasound imaging and the like.Biopsy is an effective means for definitive diagnosis, however, itplaces great burden on a patient to be diagnosed, and also is notsuitable for tracing changes with time in lesions. X-ray imaging and MRIinevitably cause exposure of a patient to be diagnosed with irradiationor electromagnetic wave. In addition, conventional imaging diagnoses asmentioned above require complicated operation and a prolonged time formeasurement and diagnosis. A large size of an apparatus also makes itdifficult to apply these methods during surgical operation.

One of reported image diagnoses includes fluorescence imaging (Lipspn R.L. et al., J. Natl. Cancer Inst., 26, 1-11 (1961)). This method employsa substance as a contrast agent that emits fluorescence upon exposure toan excitation light having a specific wavelength. The method comprisesthe step of exposing a body with an excitation light from outside thebody and then detecting fluorescence emitted from the fluorescentcontrast agent in vivo.

An example of the fluorescent contrast agent include, for example, aporphyrin compound that accumulates in tumor and is used forphotodynamic therapy (PDT), e.g., haematoporphyrin. Other examplesinclude photophyrin and benzoporphyrin (see, Lipspn R. L. et al., supra,Meng T. S. et al., SPIE, 1641, 90-98 (1992), WO84/04665 an the like).However, these compounds have phototoxicity since they are originallyused for PDT (PDT requires such property), and accordingly, thesecompounds are not desirable as diagnostic agents.

Retinal circulatory microangiography using a known fluorescent dye, suchas fluorescein, fluorescamine, and riboflabin, has been known (U.S. Pat.No. 4,945,239). However, these fluorescent dyes emit fluorescence in aregion of a visible light of 400-600 nm which only achieves lowtransmission through living tissue, and consequently, detection of alesion in a deeper part of a body is almost impossible.

Cyanine compounds including indocyanine green (hereinafter abbreviatedas “ICG”), which are used to determine liver function and cardiacoutput, have been also reported to be useful as fluorescent contrastagents (Haglund M. M. et al., Neurosurgery, 35, 930 (1994), Li, X. etal., SPIE, 2389, 789-797 (1995)). Cyanine compounds have absorbance in anear infrared light region (700 to 1300 nm).

Near infrared light has a high transmission property through livingtissues and can pass through the skull of about 10 cm, and from thesereasons, said light has been focused recently in the filed of clinicalmedicine. For example, the optical CT technique (a CT technique usingoptical transmission of a medium) has become focused as a new technologyin the clinical flied, because near infrared light can pass through aliving body and, oxygen concentration and circulation in vivo can bedetected by using a light within this region.

The cyanine compound emits fluorescence in the near infrared region, alight of which region has excellent permeability in living tissues asexplained above, and accordingly a use as a fluorescent contrast agenthas been proposed. Various cyanine compounds have been developed inrecent years, and approaches for use as fluorescent contrast agents havebeen made (WO96/17629, WP97/13490 and the like). However, an agenthaving a satisfactory distinguishing ability of a lesion from normaltissues, i.e., an agent having a satisfactory selectively to a targetsite to be imaged, has not yet been available.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a fluorescent contrastagent which emits fluorescence in the near infrared region that isexcellent in permeability in a living tissue, and enables specificimaging of tumor and/or blood vessel. Another object of the presentinvention is to provide a method of fluorescence imaging using said nearinfrared fluorescent contract agent.

The inventors of the present invention conducted various studies toachieve the foregoing objects. As a result, by introducing carboxylicacid or an aryl group to cyanine dyes, they succeeded in providing afluorescent contrast agent having high tumor selectivity. They alsosucceeded in establishing a method for fluorescence imaging by usingsaid contrast agent. The present invention was achieved on the basis ofthe above findings.

The present invention thus provides a near infrared fluorescent contrastagent comprising a compound represented by the following formula [I] ora pharmaceutically acceptable salt thereof:

wherein R¹, R², R⁷, and R⁸ independently represent a substituted orunsubstituted C₁-C₁₀ alkyl group or a substituted or unsubstituted arylgroup, and R¹ and R² and/or R⁷ and R⁸ may bind to each other to form aring; R³, R⁴, R⁵, R⁶, R⁹, R¹⁰, R¹¹, and R¹² independently represent ahydrogen atom, a substituted or unsubstituted C₁-C₆ alkyl group, asubstituted or unsubstituted aryl group, a substituted or unsubstitutedheteroaryl group, a halogen atom, cyano group, carboxyl group, or sulfogroup, and R³, R⁴, R⁵, R⁶, R⁹, R¹⁰, R¹¹, and R¹² may bind to each otherto form a ring; X¹ and X² independently represent a substituted orunsubstituted C₁-C₁₅ alkyl group or a substituted or unsubstituted arylgroup and X¹ and X² in total have 0 to 4 carboxyl groups, provided thatwhen the number of the carboxyl group is 0 or 1, each of X¹ and X² is aC₁-C₅ carboxyalkyl group or a sulfoalkyl group and at least one of R³,R⁴, R⁵, R⁶, R⁹, R¹⁰, R¹¹, and R¹² represents a substituted orunsubstituted aryl group or a substituted or unsubstituted heteroarylgroup; m¹ represents 0 or 1; m² represents 0 or 1; m³ represents 0 or 1;L¹, L², L³, L⁴, L⁵, L⁶, and L⁷ independently represent a substituted orunsubstituted methine group, provided that when two or more of themethine groups have substituents, the substituent may bind to each otherto form a ring, provided that when each of X¹ and X² has one carboxylgroup, each of X¹ and X² is carboxyl group-substituted hydrocarbon groupand at least one of the methine groups represented by L¹, L², L³, L⁴,L⁵, L⁶, and L⁷ is a substituted methine group and R⁴ and R¹⁰ represent asulfo group; M represents a hydrogen atom, a metal, or a quaternaryammonium salt; and n represents an integer of 1 to 7 necessary forneutralizing charge.

According to a preferred embodiment of the above invention, each of m¹,m², and m³ is simultaneously 1, and X¹ is a group represented by thefollowing formula (i):

wherein Y¹ and Y² independently represent a substituted or unsubstituteddivalent linking group.

According to a more preferred embodiment, X¹ and X² independentlyrepresent a group represented by the following formula (i):

wherein Y¹ and Y² independently represent a substituted or unsubstituteda divalent bond.

According to further preferred embodiment, at least one of R³, R⁴, R⁵,R⁶, R⁹, R¹⁰, R¹¹, and R¹² is a substituted or unsubstituted aryl groupor a substituted or unsubstituted heteroaryl group, and according tostill further preferred embodiment, at least one of R⁴, R⁵, R¹⁰, and R¹¹is a substituted or unsubstituted aryl group or a substituted orunsubstituted heteroaryl group; and each of X¹ and X² is independently aC₁-C₆ carboxylalkyl group or a sulfoalkyl group.

According to another preferred embodiment, X¹ and X² independentlyrepresent a group represented by the following formula:

wherein Y³ represents a C₁-C₁₀ hydrocarbon group and at least one of themethine groups represented by L¹, L², L³, L⁴, L⁵, L⁶, and L⁷ is asubstituted methine group and each of R⁴ and R¹⁰ is a sulfo group.

Preferably, the number of sulfo group in a molecule is two or less.

According to further preferred embodiment, Y¹ represents—(CH₂)_(p)CONH—wherein p represents an integer of 1 to 4 and Y₂represents —(CH₂)— or (CH₂)₂—.

The aforementioned near infrared fluorescent contrast agent maypreferably be used for tumor imaging or angiography.

From another aspect, provided is a method of fluorescence imaging whichcomprises the steps of introducing the aforementioned near infraredfluorescent contrast agent into a living body, exposing said body to anexcitation light, and detecting near infrared fluorescence from thecontrast agent.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 is a photograph showing the results of fluorescence imaging atgiven times after the administration of Compound 2 of the presentinvention.

FIG. 2 is a photograph showing the results of fluorescence imaging atgiven times after the administration of ICG as a reference.

FIG. 3 is a photograph showing the results of fluorescence imaging atgiven times after the administration of Compound A as a reference.

FIG. 4 is a schematic view of experimental set up for fluorescenceimaging in Test Example 2. In the figure, SHG represents second harmonicgeneration; THG represents third harmonic generation; and OPO representsoptical parametric oscillator.

FIG. 5 is a photograph showing the results of fluorescence imaging atgiven times after the administration of Compound 5 of the presentinvention.

FIG. 6 is a photograph showing the results of fluorescence imaging atgiven times after the administration of Compound 7 of the presentinvention.

FIG. 7 is a photograph showing the results of fluorescence imaging atgiven times after the administration of Compound 10 of the presentinvention.

FIG. 8 is a photograph showing the results of fluorescence imaging atgiven times after the administration of Compound B as a reference.

BEST MODE FOR CARRYING OUT THE INVENTION

The C₁-C₁₀ alkyl group represented by R¹, R², R⁷, and R⁸ may be linear,branched, cyclic, or a combination thereof (an alkyl group and an alkylmoiety of a functional group containing the alkyl moiety have the samemeaning in the specification unless otherwise specifically mentioned).As the unsubstituted alkyl group, for example, methyl group, ethylgroup, propyl group, butyl group, and hexyl group can be used. Thenumber, kind, or position of substituents on the substituted alkyl groupare not particularly limited. As the substituted alkyl, for example,sulfoalkyl group, carboxylalkyl group, hydroxyalkyl group, alkoxyalkylgroup, aminoalkyl group, halogenated alkyl group, cyanoalkyl group,aryl-substituted alkyl group, heteroaryl-substituted alkyl group and thelike can be used.

The aryl group represented by R¹, R², R⁷, and R⁸ may be either amonocyclic ring or a condensed ring, for example, a C₆-C₁₄ aryl group,preferably C₆-C₁₀ aryl group can be used (an aryl group and an arylmoiety of a functional group containing the aryl moiety have the samemeaning unless otherwise specifically mentioned). As the aryl group,preferably phenyl group or naphthyl group, more preferably phenyl groupmay used. As the substituted aryl group, sulfophenyl group,hydroxyphenyl group, aminophenyl group can be used.

Further, R¹ and R², R⁷ and R⁸ may bind to each other to form a ring.Examples of the ring formed include, for example, cyclopentyl ring,cyclohexyl ring and the like. R¹, R², R⁷, and R⁸ are preferably methylgroup or ethyl group, more preferably methyl group.

R³, R⁴, R⁵, R⁶, R⁹, R¹⁰, R¹¹, and R¹² independently represent a hydrogenatom, a substituted or unsubstituted C₁-C₆ alkyl group, a substituted orunsubstituted aryl group, a substituted or unsubstituted heteroarylgroup, a halogen atom, cyano group, carboxyl group, or sulfo group, andtwo adjacent groups selected from the group consisting of R³, R⁴, R⁵,and R⁶ or those selected from the group consisting of R⁹, R¹⁰, R¹¹, andR¹² may independently bind to each other to form a ring. The ring formedmay be saturated or unsaturated, and may be a hydrocarbon ring or aheterocyclic ring. For example, R³ and R⁴, R⁴ and R⁵, R⁵ and R⁶, R⁹ andR¹⁰, R¹⁰ and R¹¹, or R¹¹ and R¹² can bind to each other to form abenzene ring or an aromatic heterocyclic ring such as pyridine ring.Preferred examples include a benzene ring formed by binding of R³ andR⁴, or R⁹ and R¹⁰.

As the aryl group represented by R³, R⁴, R⁵, R⁶, R⁹, R¹⁰, R¹¹, and R¹²,for example, phenyl group or naphthyl, group can be used, as theheteroaryl group, for example, thienyl group, benzothienyl group, furylgroup, benzofuryl group, pyrrolyl group, imidazolyl group, or quinolylgroup can be used. One to four optional substituents may be present onthe aryl group and the heteroaryl group. The position of thesubstituents is not limited, and when two or more substituents arepresent, they may be same or different. As such substituents, forexample, hydroxyl group, a halogen atom such as fluorine atom, chlorineatom, bromine atom, and iodine atom; a C₁-C₆ alkyl group such as methylgroup, ethyl group, n-propyl group, isopropyl group, n-butyl group,sec-butyl group, tert-butyl group; C₁-C₆ halogenated alkyl group such astrifluoromethyl group; a C₁-C₆ alkoxyl group such as methoxy group,ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group,sec-butoxy group, tert-butoxy group; a C₁-C₆ alkylenedioxy group such asmethylenedioxy group, ethylenedioxy group; carboxyl group; a C₁-C₆alkoxycarbonyl group; an unsubstituted amino group; a C₁-C₆alkyl-substituted amino group such as methylamino group, dimethylaminogroup, ethylamino group; a sulfo group; or a cyano group and the likecan be used.

X¹ and X² independently represent a substituted or unsubstituted C₁-C₁₅alkyl group or a substituted or unsubstituted aryl group, and X¹ and X²have one to four carboxyl groups in total of X¹ and X². As theunsubstituted alkyl represented by X¹ and X², for example, methyl group,ethyl group, propyl group, butyl group, isobutyl group, sec-butyl group,tert-butyl group, pentyl group, isopentyl group, neopentyl group,tert-pentyl group, 2-methylpropyl group, or 1,1-dimethylpropyl group canbe used. The alkyl group may be linear, branched, cyclic, or acombination thereof, and a linear or branched alkyl group is preferred.

As the substituted alkyl group represented by X¹ and X², for example, asulfoalkyl group (such as 2-sulfoethyl group, 3-sulfopropyl group,3-methyl-3-sulfopropyl group, 4-sulfobutyl group and the like), acarboxyalkyl group (such as 1-carboxymethyl group, 2-carboxyethyl group,3-carboxypropyl group, 4-carboxybutyl group and the like), ahydroxyalkyl group, an alkoxyalkyl group, an aminoalkyl group, ahalogenated alkyl group, a cyanoalkyl group, a heteroaryl-substitutedalkyl group, an aryl group, or a heteroaryl group can be used. The alkylmoiety of these groups is the same as those defined in theabove-mentioned unsubstituted alkyl group. As the substituted orunsubstituted aryl group represented by R¹, R², R⁷, and R⁸, phenylgroup, sulfophenyl group, hydroxyphenyl group, or aminophenyl group canbe used.

When the number of carboxyl group of X¹ and X² is 0 or 1, a C₁-C₅carboxyalkyl group or a sulfoalkyl group can be used as the X¹ and X².

As the divalent liking group represented by Y¹ and Y², for example, asubstituted or unsubstituted C₁-C₆ alkylene group such as methylenegroup, ethylene group, n-butylene group, methylpropylene group, orphenylene group can be used. As another example, a linking grouprepresented by the following formula can be used:

wherein q represents an integer of 1 to 4, and the symbol “−” representsa bonding position. These hydrocarbon groups may have substituents andmay contain one or more hetero atoms. For example, they may contain anether bond, a thioether bond, a disulfide bond, an amide bond, an esterbond, a sulfonamide bond, or a sulfoester bond.

As the divalent linking group represented by Y¹ and Y², for example, abond represented by the following formula can also be used:

wherein p represents an integer of 1 to 4, and the symbol “−” representsa bonding position. An preferred example of Y¹ include a linking grouprepresented by the following formula:

wherein p represents an integer of 1 to 4. Most preferably, Y¹ is—(CH₂)_(p)—CO—NH— (wherein p represents an integer of 1 to 4). Preferredexamples of Y² include methylene group or ethylene group.

L¹, L², L³, L⁴, L⁵, L⁶, and L⁷ independently represent a substituted orunsubstituted methine group, wherein m¹, m², and m³ independentlyrepresent 0 or 1. It is preferred that each of m¹, m², and m³ issimultaneously 1. Examples of the substituent on the methine groupinclude a substituted or unsubstituted alkyl group, a halogen atom, asubstituted or unsubstituted aryl group, or a lower alkoxy group and thelike. An specific examples of the substituted aryl group includes4-chlorophenyl group and the like. The lower alkoxy group may preferablybe a C₁-C₆ alkoxy group which may be linear or branched. Specificexamples include methoxy group, ethoxy group, propoxy group, butoxygroup, tert-butoxy group, pentyloxy group and the like, and methoxygroup or ethoxy group is preferred. As the substituent of the methinegroup, methyl group or phenyl group can preferably be used.

When the methine groups selected from L¹, L², L³, L⁴, L⁵, L⁶, and L⁷ aresubstituted, the substituents on the methine groups may bind to eachother to form a ring. Preferably, the substituents on the methine groupsmay bind to form a ring containing three successive methine groupselected from the group consisting of L¹, L², L³, L⁴, L⁵, L⁶, and L⁷. Asan example wherein the substituents on the methine groups bind to eachother to form a ring containing three successive methine group selectedfrom the group of L¹, L², L³, L⁴, L⁵, L⁶, and L⁷ include, for example, acompound wherein 4,4-dimethylcyclohexene ring is formed to contain L³,L⁴, and L⁵. A particularly preferred example of a partial structure inwhich a conjugated methine chain formed by methine groups selected fromthe group of L¹, L², L³, L⁴, L⁵, L⁶, and L⁷ contains a ring includes agroup represented by the following general formula (a):

wherein Z represents a nonmetallic atom group necessary for forming a 5-or 6-membered ring, and A represents a hydrogen atom or a monovalentgroup.

Examples of the nonmetallic atom group necessary for forming a 5- to10-membered ring represented by Z include, for example, a carbon atom, anitrogen atom, an oxygen atom, a hydrogen atom, a sulfur atom, a halogenatom (fluorine atom, chlorine atom, bromine atom, iodine atom) and thelike. Examples of the 5- or 6-membered ring in the partial structurerepresented by the general formula (a) include, for example,cyclopentene ring, cyclohexene ring, and 4,4-dimethylhexene ring, andcyclopentene ring or cyclohexene ring is preferred.

Examples of the monovalent group represented by A include, for example,a substituted or unsubstituted alkyl group, a substituted orunsubstituted aryl group, a substituted or unsubstituted aralkyl group,a substituted or unsubstituted lower alkoxy group, a substituted orunsubstituted amino group, a substituted or unsubstitutedalkylcarbonyloxy group (such as acetoxy group), a substituted orunsubstituted alkylthio group, a substituted or unsubstituted arylthiogroup, cyano group, nitro group, a halogen atom and the like.

Specific examples of the aralkyl group represented by A include benzylgroup, 2-phenylethyl group, 3-phenylpropyl and the like. Examples of thesubstituent of the aralkyl group include, for example, sulfo group,carboxyl group, hydroxyl group, a substituted or unsubstituted alkyl,group, an alkoxy group, a halogen atom and the like. Specific examplesof the substituted amino group represented by A include, for example, analkylamino group (such as methylamino group, ethylamino group and thelike), a dialkylamino group (such as dimethyl amino group, diethylaminogroup and the like), phenylamino group, diphenylamino group,methylphenylamino group, a cyclic amino group (such as morpholino group,imidazolidino group, ethoxycarbonylpiperadino group and the like). Whenthe substituted amino group has a further substituent, sulfo group,carboxyl group and the like can be used as the substituent. Specificexamples of the arylthio group represented by A include phenylthiogroup, naphthylthio group and the like, and examples of a substituent ofthe arylthio group include sulfo group, carboxyl group and the like.

Examples of the monovalent group represented by A include phenylaminogroup, diphenylamino group, ethoxycarbonyl piperazino group, arylthiogroup and the like.

Y represents a nonmetallic atom necessary for forming a 5- to10-membered heterocyclic ring, preferably, a 5- or 6-memberedheterocyclic ring (the heterocyclic ring may be a condensed ring).Examples of the 5- to 10-membered heterocyclic ring formed by Y includethe following rings: thiazole ring (such as thiazole, 4-methylthiazoleand the like), benzothiazole ring (such as benzothiazole,4-chlorobenzothiazole and the like), naphthothiazole ring (such asnaphtho[2,1-d]-thiazole, naphtho[1,2-d]thiazole and the like),thiazoline ring (such as thiazoline, 4-methylthiaazoline and the like),oxazole ring (such as oxazole, 4-nitrooxazole and the like), benzoxazole(such as benzoxazole, 4-chrolobenzoxazole and the like), naphthoxazole(such as naphtho[2,1-d]oxazole, naphtho[1,2-d]oxazole and the like),selenazole ring (such as selenazole, 4-phenyl selenazole and the like),benzoselenazole ring (such as benzoselenazole, 4-chrolobenzoselenazole),naphtoselenazole ring (such as naphtho[2,1-d]selenazole,naphtho[1,2-d]selenazole and the like), 3,3-dialkylindolenine ring (suchas 3,3-dinitroindolenine, 3,3-diethylindolenine,3,3-dimethyl-5-nitroindolenine and the like), imidazole ring (such as1-alkylimidazole, 1-alkyl-4-phenylimidazole and the like), pyridine ring(such as 2-pyridine, 5-methyl-2-pyridine and the like), quinoline ring(such as 2-quinoline, 3-methyl-2-quinoline and the like),imidazo[4,5-b]quinoxaline ring (such as1,3-diethylimidazo[4,5-b]quinoxaline and the like) and the like.Preferred examples of the 5- to 10-membered heterocyclic ring formed byY include 3,3-dialkylindolenine ring.

M represents hydrogen atom, a metal, quaternary ammonium salt, or otherpharmaceutically acceptable salts. The “pharmaceutically acceptablesalts” may be any salt which can form nontoxic salts with the compoundrepresented by the general formula [I]. Examples include, for example,alkaline metal salt such as a sodium salt, a potassium salt and thelike; alkaline-earth metal salt such as a magnesium salt, a calcium saltand the like; organic ammonium salt such as a ammonium salt, a triethylammonium salt, tributyl ammonium salt, pyridinium salt and the like;salt of amino acid such as lysine salt, arginine salt and the like.Particularly preferred is a sodium salt with a reduced toxicity to aliving body.

The compound of the present invention may have one or more asymmetriccarbon atoms depending on the kind of substituents. Sulfur atoms may actas asymmetric center. Any optical isomers in an optically pure formbased on one or more asymmetric carbon atoms, any mixture of the aboveoptical isomers, racemates, diastereomers based on two or moreasymmetric carbon atoms, any mixture of the above diastereomers and thelike fall within the scope of the present invention.

Specific examples of the compound of the present invention are shownbelow. However, the scope of the present invention is not limited by thefollowing compounds.

The cyanine dye represented by the formula [I] or [II] can besynthesized according to known preparation methods of cyanine dyecompounds, for example, those disclosed in the Cyanine Dyes and RelatedCompounds, F. M. Hamer, John Wiley and Sons, New York, 1964, Cytometry,11, 416-430 (1990), Cytometry, 12, 723-730 (1990), Bioconjugate Chem, 4,105-111 (1993), Anal. Biochem., 217, 197-204 (1994), Tetrahedron, 45,4845-4866 (1989), EP-A-0591820A1, EP-A-0580145A1 and the like.Alternatively, they can be semisynthesized from a commercially availablecyanine dye compound by known methods. More specifically, they can besynthesized by reacting a diaryl compound with a heterocyclic quaternarysalt.

The methods for preparing the cyanine dye compounds represented by theabove formula [I] or [II] are not particularly limited, and thecompounds can be synthesized by various synthetic routes. Specificpreparations of typical compounds of the present invention are disclosedin the Examples of the specification. Accordingly, one of ordinary skillin the art can prepare the cyanine dye compounds that falls within thescope of the above general formulas by referring to the methodsdescribed in the Examples, and if necessary, by adding appropriatealteration or modification to the methods and by appropriately choosingstarting materials and reagents. For the preparation, a reactionselected from various reactions such as condensation, addition,oxidation, reduction and the like may be employed alone or incombination. These reactions are explained in detail in the literature.For example, various methods or material compounds described as unitsynthetic operations in “Jikken Kagaku Kouza” (published by Maruzen,Ltd., each separate volume contained in the first to forth comprehensiveedition is available) can be suitably used. In addition, syntheses ofthe compounds of the present invention are specifically described in thespecification of PCT/JP01/06689, whose disclosures are hereinincorporated by reference.

For example, where the above defined functional groups may change in areaction step or they are not suitable to conduct a reaction step in thepreparation, a desirable step can be sometimes conducted efficiently byutilizing various methods which are conventionally used in the filed oforganic synthetic chemistry, for example, means for protection ordeprotection of functional groups, or treatments such as oxidation,reduction, hydrolysis and the like. Synthetic intermediate compounds andthe target compounds in the above steps can be isolated and purified byconventional purification methods used in organic synthetic chemistrysuch as filtration, extraction, washing, drying, concentration,recrystallization, and various chromatography and the like. Thesynthetic intermediate products can be used in the next reaction withoutisolation.

As the active ingredient of the near infrared fluorescent contrast agentof the present invention, the compound represented by the generalformula [I] in or [II] or a salt thereof may be used alone or incombination. More specifically, the active ingredient may be containedin the contrast agent in a form of a suspension or a solution in asolvent such as injectable distilled water, physiological saline,Ringer's solution and the like. Additives such as pharmaceuticallyacceptable carrier, excipients and the like may also be formulated, ifdesired. Examples of these additives include substances such aspharmaceutically acceptable electrolytic solutions, buffering solutions,detergents, and substances for adjusting osmotic pressure, substancesfor improving stability or solubility such as cyclodextrin, liposome andthe like. Any additives ordinarily available in the art may be used. Thenear infrared fluorescent contrast agent of the present invention ispreferably synthesized through sterilization processes when used as amedicament for clinical application.

The contract agent can be administered to a living body by injection,spraying, or topical application such as intravascular application(venous, arterial), oral application, intraperitoneal application,percutaneous application, subcutaneous application, intracysticalapplication, or intrabronchial application. Preferably, the contrastagent may be administered into blood vessels in the form of an aqueoussolution, an emulsion or a suspension.

The dose of the near infrared fluorescent contrast agent of the presentinvention is not particularly limited insofar as the dose enablesdetection of a site to be diagnosed. The dose may appropriately beincreased or decreased depending on the type of the compound to be usedthat emits near infrared fluorescence, the age, body weight and a targetorgan of a subjects to be administered and the like. Typically, the doseas the weight of the compound may be 0.1 to 100 mg/kg body weight,preferably 0.5 to 20 mg/kg body weight.

The contrast agent of the present invention may also be appropriatelyused for various animals other than human. A formulation foradministration, the route of administration, a dose and the like may beappropriately chosen depending on the body weight and conditions of thetarget animals.

The compounds of the present invention represented by the above formula[I] and [II] have property to highly accumulated in tumor tissues.Utilizing said property, the present invention also provides thefluorescent contrast agent which enables specific imaging of a tumortissue. In addition, the class of the compounds of the present inventionhave long-term retention in blood vessels, and therefore, thefluorescent contrast agent of the present invention is also useful forangiography.

The method for fluorescence imaging of the present invention ischaracterized by the use of the near infrared fluorescent contrast agentof the present invention. The method for imaging can be carried out byone of ordinary skill in the art according to known methods, and each ofparameters such as excitation wavelength and fluorescence wavelength tobe detected may appropriately be determined to achieve optimal imagingand evaluation, depending on the kind of near infrared fluorescencecontrast agent to be administered and a subject to be administered. Theperiod of time from administration of the near infrared fluorescentcontrast agent of the present invention to the start of fluorescenceimaging according to the present invention may vary depending on thekind of the near infrared fluorescent contrast agent to be used and asubject to be administered. For example, when the contrast agentcomprising a compound of the formula [I] or formula [II] is administeredfor tumor imaging, a lapse time may be about 10 minutes to 24 hoursafter administration. When the lapse time is too short, fluorescencefrom every site may be still too intense and the target site is notdistinguishable from other sites, and when the lapse time is too long,the contrast agent may be excreted from the body. When imaging of bloodvessel is desired, the compound of the formula [I] or formula [II] isdetected immediately after administration or in about 30 minutes afterthe administration.

For example, the fluorescence imaging can be conducted by the followingsteps. A near infrared fluorescent contrast agent of the presentinvention is administered to a subject to be diagnosed, and then thesubject is exposed to an excitation light using an apparatus generatingexcitation light. Then, fluorescence from the near infrared fluorescentcontrast agent, which is generated by the excitation light, is detectedby using a fluorescence detector. The wavelength for excitation variesdepending on the type of the near infrared fluorescent contrast agent tobe used, and is not limited as long as the compounds efficiently emitsfluorescence in the near infrared region. Preferably, a near infraredlight having superior bio-permeability may be used. The wavelength ofthe near infrared fluorescence to be detected also varies depending onthe contrast agent to be used. In general, an excitation light having awavelength of 600 to 1000 nm, preferably 700 to 850 nm, may be used andnear infrared fluorescence having a wavelength of 700 to 1000 nm,preferably, 750 to 900 nm, may be detected. As the apparatus forgenerating the excitation light, a conventional excitation light sourcesuch as various lasers (e.g., ion lased, dye laser and semiconductorlaser), halogen light source, xenon light source and the like may beused. Various optical filters may be used to obtain optimal excitationwavelength, if desired. For detection of fluorescence, various opticalfilters may be used for selection of the fluorescence generated from thenear infrared fluorescent contrast agent.

The detected fluorescence is data-processed as fluorescence informationto construct fluorescence images to be recorded. Examples of the methodfor preparation of fluorescence images include, for example, a methodcomprising the step of irradiating the target tissue in a wide range,detecting fluorescence with a CCD camera, and then image-processing thefluorescence information obtained; a method using an optical CT device;a method using an endoscope; or a method using fundus oculi camera andthe like.

According to the fluorescence imaging method of the present invention,systemic diseases, tumors, blood vessels and the like can be visualizedwithout damaging a living body.

EXAMPLES

The present invention will be more specifically explained by referringto synthetic examples and a test example. However, the scope of thepresent invention is not limited to the following examples. In theexamples, the serial numbers of the compounds correspond to that of thecompounds listed in the above with chemical structures.

Example 1 Synthesis of Compound 1, Compound 2, and Compound 3

Synthetic route of Compound 1 is shown below.

Synthesis of Intermediate 1

The starting material 1 (20.9 g, 0.1 mol), 2-bromopropinic acid (23.0 g,0.15 mol), and o-dichlorobenzene (20 ml) were heated and stirred at 140°C. for 2 hours. After the reaction was completed, the reaction mixturewas added with acetone (200 ml) and cooled to room temperature, and thenthe resultant crystal was filtrated to obtain Intermediate 1 (20.3 g,yield: 56%)

Synthesis of Intermediate 2

The Intermediate 1 obtained above (10.0 g, 28 mmol) and1,7-diaza-1,7-diphenyl-1,3,5-heptatriene hydrochloride (3.9 g, 14 mmol)were dissolved in acetonitrile (70 ml) and water (11 ml), and theresulting solution was added with triethylamine (8.4 g, 91 mmol) andacetic anhydride (8.5 g, 91 mmol) and the mixture was stirred at roomtemperature for overnight. The reaction mixture was added to 0.1Nhydrochloric acid (900 ml) dropwise and the crystals precipitated werefiltrated. The crystal was purified by column chromatography (eluent:methylene chloride:methanol=95:5˜90:10) to obtain Intermediate 2 (2.1 g,yield: 12%)

Synthesis of Intermediate 3

The Intermediate 2 obtained above (21.0 g, 1.5 mmol), L-asparticacid-di-t-butylester monohydrate (1.3 g, 4.5 mmol),4-dimethylaminopyridine (40 mg, 0.3 mmol) were dissolved in methylenechloride (50 ml) and the solution was cooled on ice. The resultantsolution was added with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) (700 mg, 4 mmol) and triethylamine (340 mg, 3 mmol),and stirred at 4° C. for overnight. The reaction mixture was added withmethylene chloride (200 ml) and 1N hydrochloric acid (200 ml), and thenthe methylene chloride layer is extracted and washed with saturatedsodium chloride solution (200 ml) and dried over sodium sulfate. Thesolvent was evaporated under reduced pressure and purified by columnchromatography (eluent: ethyl acetate:methanol=95:5 to 80:20) to obtainIntermediate 3 (1.1 g, yield: 64%)

Synthesis of Compound 1, Compound 2, and Compound 3

Intermediate 3 (500 mg, 0.5 mmol) was dissolved in trifluoroacetic acid(5 ml) and reacted at 4° C. for overnight, and then trifluoroacetic acidwas evaporated under reduced pressure. The resulting residue was addedwith water (50 ml) and then the resulting crystals were collected byfiltration and washed with water and ethyl acetate to obtain Compound 1(390 mg, yield: 90%).

Compound 1 was purified by column chromatography using Sephadex (LH-20,Pharmacia) (eluent: methanol) to obtain Compound 2.

Compound 1 was applied to an ion exchange resin column CR 11 (MitsubishiChemical, Co., Ltd.) to obtain Compound 3.

Compound 1

¹H-NMR (CD₃OD) δ 1.98 (s, 12H), 2.70 (d, J=7.2 Hz, 4H), 2.80 (t, J=7.2Hz, 4H), 3.30 (MeOH), 4.50 (t, J=7.2 Hz, 4H), 4.60 (t, J=7.2 Hz, 2H),4.80 (H₂O), 6.40 (d, J=13.2 Hz, 2H), 6.63 (dd, J=13.2, 13.2 Hz, 2H),7.40-7.50 (m, 2H), 7.58-7.66 (m, 5H), 7.95-8.07 (m, 6H), 8.20 (d, J=7.2Hz, 2H)

Compound 2

¹H-NMR (CD₃OD) δ 1.99 (s, 12H), 2.72 (d, J=7.2 Hz, 4H), 2.80 (t, J=7.2Hz, 4H), 3.30 (MeOH), 4.50 (t, J=7.2 Hz, 4H), 4.60 (t, J=7.2 Hz, 2H),4.80 (H₂O), 6.38 (d, J=13.2 Hz, 2H), 6.61 (dd, J=13.2, 13.2 Hz, 2H),7.40-7.50 (m, 2H), 7.58-7.67 (m, 5H), 7.96-8.07 (m, 6H), 8.21 (d, J=7.2Hz, 2H)

Compound 3

¹H-NMR (CD₃OD) δ 1.98 (s, 12H), 2.56-2.65 (m, 4H), 2.75-2.85 (m, 4H),3.30 (MeOH), 4.45-4.50 (m, 4H), 4.80 (H₂O), 6.20 (d, J=13.2 Hz, 2H),6.65 (dc, J=13.2, 13.2 Hz, 2H), 7.40-7.50 (m, 2H), 7.58-7.70 (m, 5H),7.95-8.07 (m, 6H), 8.20 (d, J=7.2 Hz, 2H)

Example 2 Synthesis of Compound 5

Compound 5 was synthesized from Intermediate 1 and1,7-diaza-5-methyl-1,7-diphenyl-1,3,5-heptatriene monohydrate in asimilar manner to that for Compound 1.

¹H-NMR (CD₃OD) δ 2.00 (s, 12H), 2.44 (s, 3H), 2.73 (d, J=7.2 Hz, 4H),2.82 (t, J=7.2 Hz, 4H), 3.31 (MeOH), 4.50 (t, J=7.2 Hz, 4H), 4.69 (t,J=7.2 Hz, 2H), 4.88 (H₂O), 6.41 (d, J=13.2 Hz, 2H), 6.65 (d, J=13.2 Hz,2H), 7.43-7.50 (m, 2H), 7.58-7.67 (m, 4H), 7.95-8.05 (m, 4H), 8.10-8.27(m, 4H)

Example 3 Synthesis of Compound 6

Compound 6 was synthesized from Intermediate 1 and1,7-diaza-5-methyl-1,7-diphenyl-1,3,5-heptatriene monohydrate in asimilar manner to that for Compound 1 except that L-glutamicacid-di-t-butylester monohydrate was used instead of L-asparticacid-di-t-butylester monohydrate.

¹H-NMR (CD₃OD) δ 1.80-2.15 (m, 4H), 2.01 (s, 12H), 2.28 (t, J=7.2 Hz,4H), 2.44 (s, 3H), 2.82 (t, J=7.2 Hz, 4H), 3.31 (MeOH), 4.40-4.50 (m,2H), 4.51 (t, J=7.2 Hz, 4H), 4.88 (H₂O), 6.42 (d, J=13.2 Hz, 2H), 6.65(d, J=13.2 Hz, 2H), 7.42-7.50 (m, 2H), 7.57-7.67 (m, 4H), 7.95-8.05 (m,4H), 8.10-8.27 (m, 4H)

Example 4 Synthesis of Compound 7

Compound 7 was synthesized from 2,3,3-trimethylindolenine in a similarmanner to that for Compound 1.

1H-NMR (CD₃OD) δ 1.70 (s, 12H), 2.05-2.13 (m, 4H), 2.55 (t, J=7.2 Hz,4H), 2.78-2.92 (m, 4H), 3.30 (MeOH), 4.10 (t, J=7.2 Hz, 4H), 4.89 (H₂O),6.45 (d, J=138.2 Hz, 2H), 6.50 (J=13.2 Hz, 2H), 7.29-7.50 (m, 8H), 7.92(dd, J=13.2, 13.2 Hz, 2H)

Example 5 Synthesis of Compound 8

Compound 8 was synthesized from 2,3,3-trimethylindolenine in a similarmanner to that for Compound 1 except that1,7-diaza-5-methyl-1,7-diphenyl-1,3,5-heptatriene monohydrochloride wasused instead of 1,7-diaza-1,7-diphenyl-1,3,5-heptatriene monohydrate.

¹H-NMR (CD₃OD) δ 1.70 (s, 12H), 1.72-1.90 (m, 8H), 2.35-2.39 (m, 7H),2.73-2.84 (m, 4H), 3.30 (MeOH), 4.08 (t, J=7.2 Hz, 4H), 4.66 (t, J=7.2Hz, 2H), 4.89 (H₂O), 6.33 (d, J=13.2 Hz, 2H), 6.63 (d, J=13.2 Hz, 2H),7.18-7.50 (m, 5H), 8.05 (dd, J=13.2, 13.2 Hz, 2H)

Example 6 Synthesis of Compound 9

Compound 9 was synthesized from 6-phenyl-2,3,3-trimethylindolenine(synthesized by a method described in the specification of the U.S. Pat.No. 6,004,536) in a similar manner to that for Compound 1.

¹H-NMR (CD₃OD) δ 1.75 (s, 12H), 2.05-2.15 (m, 4H), 2.45-2.55 (m, 4H),2.75-2.84 (m, 4H), 3.30 (MeOH), 4.20 (t, J=7.2 Hz, 4H), 4.80 (H₂O), 6.38(J=13.2 Hz, 2H), 6.62 (J=13.2 Hz, 2H), 7.43-7.70 (m, 17H), 7.95 (dd,J=13.2, 13.2 Hz, 2H)

Example 7 Synthesis of Compound 10

Compound 10 was synthesized from 6-bromo-2,3,3-trimethyl-indolenine in asimilar manner to that for Compound 1.

¹H-NMR (CD₃OD) δ 1.68 (s, 12H), 2.00-2.15 (m, 4H), 2.40-2.55 (m, 4H),2.77-2.92 (m, 4H), 3.30 (MeOH), 4.08 (t, J=7.2 Hz, 4H), 4.82 (m, 2H),6.38 (J=13.2 Hz, 2H), 6.65 (J=13.2 Hz, 2H), 7.30-7.40 (m, 4H), 7.50-7.72(m, 3H), 7.90-8.02 (m, 2H)

Example 8 Synthesis of Compound 11

Compound 11 was synthesized from 5-phenyl-2,3,3-trimethyl-indolenine ina similar method to that for Compound 1.

¹H-NMR (CD₃OD) δ 1.78 (s, 12H), 2.39 (s, 3H), 2.70-2.84 (m, 8H), 3.30(MeOH), 4.30-4.46 (m, 4H), 4.60-4.68 (m, 2H), 6.39 (J=13.2 Hz, 2H), 6.66(J=13.2 Hz, 2H), 7.30-7.48 (m, 9H), 7.56-7.72 (m, 3H), 8.05 (J=13.2 Hz,13.2 Hz)

Example 9 Synthesis of Compound 13 and Compound 14

Synthetic route of Compound 13 and Compound 14 is shown below.

An intermediate compound (375 mg), which was obtained by reacting5-sulfo-2,3,3-trimethylindolenine (prepared according to the methoddescribed in the Japanese Patent Unexamined Publication (KOKAI) No.(Hei)2-233658) and 1,7-diaza-1,7-diphenyl-1,3,5-heptatrienemonohydrochloride in methanol in the presence of triethylamine andacetic anhydride, was dissolved in 5 ml of methanol, and then applied toan column filled with cationic ion exchange resin IRC-50 (Organo,eluent: methanol). The solvent was evaporated to give the proton form ofthe carboxylic acid. The resulting product was dissolved in 3 ml ofdimethylformamide, and the solution was added with 338 mg (1.2 mmol) ofdibutyl aspartate hydrochloride, 24 mg (0.2 mmol) ofdimethylaminopyridine, and 121 mg (1.2 mmol) of triethylamine, and thenthe mixture was cooled on ice bath. The mixture was added with 230 mg (2mmol) of hydroxysuccinimide (HOSI) and 288 mg (1.4 mmol) ofN,N-dicyclohexyl-carbodiimide (DCC), and the resulting mixture wasstirred overnight. The reaction mixture was added with 200 ml of a mixedsolvent of ethyl acetate/hexane (1:1) and crystals precipitated werecollected by filtration. The crystals were purified by columnchromatography (eluent: methylene chloride:methanol=10:1 to 2:1) toobtain diamide compound (135 mg) and monoamide compound (94 mg).

Each of the resulting diamide compound (120 mg) and monoamide compound(60 mg) was dissolved in 2 ml of trifluoroacetic acid, and then themixture was stirred at room temperature for 1 hour. The reaction mixturewas dissolved in water/methanol (1/1(v/v)) and purified by columnchromatography using Sephadex (LH-20, Pharmacia, eluent: methanol). Theresulting crystals were dissolved in a small volume of methanol, and thesolution was added with a saturate solution of potassium acetate inmethanol. Crystals precipitated were collected by filtration to obtainCompound 13 (35 mg, yield 7%) and Compound 14 (15 mg, yield 5%).

Compound 13

¹H-NMR (D₂O) δ 1.73 (s, 12H), 2.50-2.65 (m, 4H), 2.68-2.73 (m, 4H),4.28-4.38 (m, 4H), 4.39-4.50 (m, 2H), 4.90 (D₂O), 6.47 (d, J=13.2 Hz,2H), 6.74 (t, J=13.2 Hz, 2H), 7.40-7.50 (m, 2H), 7.60 (t, J=13.2 Hz,1H), 7.80-8.05 (m, 6H)

Compound 14

¹H-NMR (D₂O) δ 1.65 (s, 6H), 1.70 (s, OH), 2.40 (d, J=7.2 Hz, 2H), 2.58(t, J=7.2 Hz, 2H), 2.70 (t, J=7.2 Hz, 2H), 4.18-4.30 (m, 4H), 4.90(D₂O), 6.18 (d, J=13.2 Hz, 1H), 6.34 (d, J=13.2 Hz, 1H), 6.48-6.62 (m,2H), 7.20 (d, J=7.2 Hz, 1H), 7.30 (d, J=57.2 Hz, 1H), 7.48 (t, J=13.2Hz, 1H), 7.68-7.95 (m, 6H)

Example 10 Synthesis of Compound 15

Synthetic route of Compound 15 is shown below.

The starting material (41.8 g, 0.2 mol) was dissolved in conc. sulfuricacid (156 ml, 2.9 mol) and reacted at 140° C. for 1 hour, and then themixture was cooled to 80° C. After the resulting solution was added toice water (300 ml), a solution obtained by dissolving sodium hydroxide(96.6 g, 2.4 mol) in water (100 ml) was carefully added to the mixture.The crystals precipitated were collected by filtration and washed withwater (120 ml). The resulting crude crystal was added with water (300ml) and methanol (100 ml), and the mixture was refluxed under stirringfor 30 minutes, and then cooled to room temperature. The resultingcrystals were collected by filtration and washed with water (100 ml) andmethanol (120 ml) to obtain Intermediate 5 (37.9 g, yield: 66%).

Compound 15 was obtained form Intermediate 5 in a similar method to thatfor Compound 13.

¹H-NMR (CD₃OD) δ 2.00 (s, 12H), 2.72 (d, J=7.2 Hz, 4H), 2.82 (t, J=7.2Hz, 4H), 3.30 (MeOH), 4.58 (t, J=7.2 Hz, 4H), 4.70 (t, J=7.2 Hz, 4H),4.86 (H₂O), 6.42 (d, J=13.2 Hz, 2H), 6.62 (dd, J=13.2, 13.2 Hz, 2H),7.62-7.70 (m, 3H), 7.95-8.12 (m, 6H), 8.28 (d, J=7.2 Hz, 2H), 8.42 (s,2H)

Example 11 Synthesis of Compound 23

Synthetic route of Compound 23 is shown below.

Synthesis of Intermediate 6

5-Sulfo-2,3,3-trimethylindolenine (synthesized according to the methoddescribed in Japanese Patent Unexamined Publication (KOKAI) No. (Hei)2-233658) (24.0 g, 0.1 mol), 2-bromopropionic acid (23.0 g, 0.15 mol)and triethylamine (10.1 g, 0.1 mol) were heated and stirred at 160° C.for 6 hours. After the reaction was completed, the reaction mixture wasadded with methanol (200 ml) and cooled to room temperature, and thenthe resulting crystals were collected by filtration to obtainIntermediate 6 (6.0 g, yield: 19.3%).

Synthesis of Compound 23

The Intermediate 1 (3.1 g, 10 mmol) obtained above and1,7-diaza-1,7-diphenyl-4-methyl-1,3,5-heptatriene monohydrochloride(Japanese Patent Unexamined Publication (KOKAI) No. (Hei) 8-295658) (1.5g, 5 mmol) were dissolved in methanol (20 ml), and the resultingsolution was added with triethylamine (2.5 g, 25 mmol) and aceticanhydride (4.6 g, 45 mmol) and the mixture was stirred at roomtemperature for 3 hours. The reaction mixture was added with sodiumacetate (3.3 g, 33 mmol) and stirred at room temperature for 30 minutes.The resulting crystals were collected by filtration and washed withmethanol (20 ml) to obtain Compound 23 (2.0 g, yield: 50.0%).

¹H-NMR (D₂O) a (ppm) 1.60 (s, 12H), 2.30 (s, 3H), 2.60 (t, 4H, J=7.2Hz), 4.20 (t, 4H, J=7.2 Hz), 6.25 (d, 2H, J=14.5 Hz), 6.55 (dd, 2H,14.5, 14.5 Hz), 7.25 (d, 2H, J=7.0 Hz), 7.70-7.80 (m, 4H), 8.00 (dd, 2H,J=14.5, 14.5 Hz)

Example 12 Synthesis of Compound 25 and Compound 26

The synthetic route of Compound 25 and Compound 26 is shown below.

Synthesis of Intermediate 7

Intermediate 7 was synthesized from 5-sulfo-2,3,3-trimethylindolenineand bromoacetic acid in a similar method to that for Intermediate 6(16.6 g).

Synthesis of Compound 25

Compound 25 was synthesized from Intermediate 7 and Intermediate 8(obtained according to the method described in Zh. Org. Khim., 13, pp.1189-1192, 1977) in a similar method to that for Compound 23 (15.0 g).

MS (FAB-, Glycerin) m/z=844

Synthesis of Compound 26

Compound 25 (4.2 g, 5 mmol) and triethylamine (1.0 g) was added to water(20 ml) and then the obtained solution was added with o-mercaptobenzoicacid (0.93 g, 6 mmol) and stirred at room temperature for 1 hour. Theobtained mixture was added with potassium acetate (2.0 g, 20 mmol), andthen added with ethanol (20 ml), the resultant crystal was filtered toobtain Compound 26 (1.3 g, yield: 27%)

MS (FAB-, Glycerin) m/z=962

Example 13 Synthesis of Compound 32

Synthetic route of Compound 32 is shown below.

Synthesis of Intermediate 9

4-Bromophenylhydrozine monohydrochloride (73.8 g, 0.33 mmol) and3-methyl-2-butanone (33.2 g, 0.40 mmol) were dissolved to ethanol (450ml) and the resulting solution was added with cone sulfuric acid (7.5ml) and refluxed under stirring for 8 hours. After the mixture wascooled to room temperature, the solution was concentrated to 100 mlunder reduced pressure. To the residue, water (400 ml) and ethyl acetate(400 ml) were added, and then pH of the aqueous layer was adjusted to 7to 8 with sodium hydroxide solution. The resulting solution wasextracted with ethyl acetate, washed with saturated sodium chloridesolution, and dried over anhydrous sodium sulfate. The resulting residuewas purified by silica gel column chromatography (eluent: hexane:ethylacetate-5:1 to 1:1) to obtain Intermediate 9 as a brown liquid (58.6 g,yield 76%)

Synthesis of Intermediate 10

Intermediate 9 (4.76 g, 20 mmol) and thiophene boronic acid (3.84 g, 30mmol) are added to dimethyl formamide (50 ml) and the resulting solutionwas added with palladium tetraxis phenylphosphine (1.16 g, 9 mmol) andcesium chloride (13.3 g, 40 mmol) and heated and stirred under nitrogenatmosphere at 100° C. for 4 hours. After water (200 ml) was added, themixture was extracted with ethyl acetate (200 ml) and washed withsaturated sodium chloride solution, and then the organic layer was driedover anhydrous sodium sulfate and evaporated under reduced pressure. Theresidue was purified by silica gel column chromatography (eluent:hexane:ethyl acetate=2:1 to 1:1) to obtain Intermediate 10 as a brownsolid (2.8 g, yield: 58%).

Synthesis of Intermediate 11

Intermediate 10 (1.40 g, 6 mmol) and triethylamine (0.59 g, 6 mmol) areadded to dimethyl formamide (3 ml), and the mixture was added dropwisewith 2-chloroethane sulfonylchloride (1.42 g, 9 mmol) under ice coolingAfter stirring was continued at room temperature for 30 minutes, thesolution was added with a solution obtained by dissolving sodiumhydroxide (0.23 g, 6 mmol) to water (2 ml) and further stirred at roomtemperature for 1 hour. To the mixture, ethyl acetate was added, and theupper layer was removed by decantation. The residue was dried to obtainIntermediate 11. The Intermediate 11 was used in the next reactionwithout further purification.

Synthesis of Compound 32

The Intermediate 11 obtained above and1,7-diaza-1,7-diphenyl-1,3,5-heptatriene monohydrochloride weredissolved in methanol (5 ml) and the resulting solution was added withtriethylamine (160 mg, 2 mmol) and anhydrous acetic acid (230 mg, 2mmol), and then the mixture was stirred at room temperature for 7 hours.This mixture was added with ethyl acetate (20 ml) and the crystalsprecipitated were collected by filtration and washed with ethyl acetate(10 ml). This crystals were dissolved in methanol (10 ml) and then thesolution was added with a saturated solution of potassium acetate inmethanol (10 ml). The crystals precipitated were collected by filtrationand washed with methanol (5 ml). The crystals were purified by SephadexLH-20 (diluent:methanol) to obtain Compound 32 (15 mg, yield: 2% (fromIntermediate 2).

¹H-NMR (CD₃OD) δ (ppm) 1.75 (s, 12H), 3.25 (t, 4H, J=7.2 Hz), 4.50 (t,4H, J=7.2 Hz), 6.40 (d, 2H, J=14.5 Hz), 6.63 (dd, 2H, 14.5, 14.5 Hz),7.07-7.12 (m, 2H), 7.33-7.45 (m, 6H), 7.53-7.75 (m, 5H), 7.96 (dd, 2H,J=14.5, 14.5 Hz)

MS (FAB-, Glycerin) m/z=760

Example 14 Synthesis Compound 33

The synthetic route of compound 33 is shown below.

Synthesis of Intermediate 12

Intermediate 12 was synthesized from Intermediate 9 and dihydroxyphenylborane in a similar method of that for Intermediate 10 (3.6 g, yield:77%).

Synthesis of Intermediate 13

Intermediate 12 (1.40 g, 6 mmol) and 1,4-butanesaltone (1.22 g, 9 mmol)were dissolved in dimethyl acetamide (2 ml) and the solution was stirredat 135° C. for 5 hours. The solution was added with ethyl acetate (20ml) and cooled to room temperature, and then the crystals precipitatedwere filtered and washed with ethyl acetate to obtain Intermediate 13(10 ml) (1.84 g, yield: 84%).

Synthesis of Compound 33

Intermediate 13 (1110 mg, 3 mmol) and1,7-diaza-1,7-diphenyl-1,3,5-heptatriene monohydrochloride (285 mg, 1mmol) were dissolved in methanol (5 ml), and the resulting solution wasadded with triethylamine (480 mg, 5 mmol) and anhydrous acetic acid (670mg, 7 mmol) and then stirred at room temperature for 7 hours. Ethylacetate (10 ml) was added to the reaction mixture and crystalsprecipitated were collected by filtration and washed with ethyl acetate(10 ml). The crystals were dissolved in methanol (5 ml) and added with asaturated solution of potassium acetate in methanol (10 ml), and thecrystals precipitated were filtered and washed with 5 ml. The crystalwas purified by Sephadex LH-20 (diluent; methanol) to obtain Compound 33(250 mg, yield: 30%).

¹H-NMR (CD₃OD) δ (ppm) 1.80 (s, 12H), 1.95-2.05 (m, 8H), 2.90 (t, 4H,J=7.2 Hz), 4.20 (t, 4H, J=7.2 Hz), 6.38 (d, 2H, J=14.5 Hz), 6.62 (dd,2H, 14.5, 14.5 Hz), 7.30-7.48 (m, 8H), 7.60-7.74 (m, 9H), 7.93 (dd, 21,J=14.5, 14.5 Hz)

MS (FAB-, Nitrobenzylalcohol) m/z=803

Example 15 Synthesis of Compound 34

Compound 34 was synthesized from Intermediate 9 and 4-methylmercaptophenyl boronic acid in a similar method to that for Compound 33(15 mg). ¹H-NMR (CD₃OD) δ (ppm) 1.68 (s, 12H), 1.95-2.10 (m, 8H), 2.50(s, 6H), 3.00 (t, 4H, J=7.2 Hz), 4.10 (t, 4H, J=7.2 Hz), 6.30 (d, 2H,J=14.5 Hz), 6.62 (dd, 2H, 14.5, 14.5 Hz), 7.20-7.70 (m, 19H)

Example 16 Synthesis of Compound 35

Synthetic route of Compound 35 is shown below.

Synthesis of Intermediate 14

25.0 g of 3-aminodiphenyl (0.15 mol) was added to 100 ml oftrifluoroacetic acid, and the mixture was cooled to the internaltemperature of 0° C. The mixture was added dropwise with a solutionobtained by dissolving 10.2 g of sodium nitrite (0.15 mol) in 100 ml ofwater while the temperature of the reaction mixture was kept below 5° C.After the dropwise addition was completed, the mixture was stirred atthe same temperature for 15 minutes, and then the mixture was added witha solution obtained by dissolving 100 g of stannic chloride (0.54 mol)in 50 ml of concentrated hydrochloric acid while the temperature of thereaction mixture was kept below 10° C. After the completion of thedropwise addition, the crystals precipitated by addition of 250 ml ofwater were collected by filtration and washed with 200 ml of methylenechloride. The resulting Intermediate 14 was dried and used for thesynthesis of Intermediate 15 without purification.

Synthesis of Intermediate 15

The above-obtained Intermediate 14 (whole amount) and 12.9 g of3-methyl-2-butanone (0.15 mol) were added to 140 ml of acetic acid, andthe mixture was heated under stirring for 2 hours and 30 minutes. Afterthe mixture was cooled to room temperature, the crystals precipitatedwere removed by filtration, and the filtrate was concentrated underreduced pressure to one quarter volume. The residue was added with 300ml of water and 300 ml of ethyl acetate, and insoluble precipitates wereremoved by filtration using celite. The filtrate was extracted withethyl acetate (300 ml, 200 ml×2), and the extract was washed with asaturated sodium hydrogen carbonate solution and saturated brine, andthen dried over sodium sulfate and the solvent was evaporated underreduced pressure. The resulting residue was purified by silica gelchromatography (eluent: hexane:ethyl acetate=3:1 to 2:1). The crystalobtained was recrystallized from 50 ml of hexane to obtain Intermediate15. 1.3 g (yield: 4%)

Synthesis of Compound 35

Compound 35 was synthesized from Intermediate 15 in a similar method tothat for Intermediate 13 and Compound 33 (65 mg).

MS (FAB-, Glycerin) m/z=842,804

¹H-NMR (D₂O) δ (ppm) 1.70 (s, 12H), 1.90-2.00 (m, 8H), 2.90 (t, 4H,J=7.2 Hz), 4.10 (t, 4H, J=7.2 Hz), 6.22 (d, 2H, J=14.5 Hz), 6.55 (dd,2H, 14.5, 14.5 Hz), 7.30-7.60 (m, 17H), 7.77 (dd, 2H, J=14.5, 14.5 Hz)

Test Example 1 Fluorescence Imaging Test

Tumor tissue pieces of mouse colon carcinoma (colon 26 carcinoma) weresubcutaneously grafted to the left breast of BALB/c nude mice (5 weeksold, Clea Japan, Inc.). Ten days later when the tumor grew to a diameterof about 8 mm, the mice were subjected to the test. As a fluorescenceexcitation light source, a titanium sapphire laser was used. The testmice were uniformly exposed to the laser light using a ring type lightguide (Sumita Optical Glass Co.) wherein dispersion of irradiation waswithin 10%. The irradiation power output was adjusted so that the powerwas about 40 μW/cm² near skin surface of the mice. The fluorescence wasexcited at the maximum excitation wavelength of each compound andfluorescence emission from the mice was detected and photographedthrough a short wavelength cutoff filter (IR84, IR8, IR88, Fuji PhotoFilm CO., LTD.) with CCD camera (C4880, Hamamatsu Photonics K.K). Thecutoff filter was selected to fit the excitation wavelength of thecompound. The exposure time was adjusted depending on the fluorescenceintensity of each compound. Compound 2 as a test compound (0.5 mg/ml)was dissolved in physiological saline or phosphate buffer (pH7.4) andadministered to the mice via a tail vein at the dose of 5.0 mg/Kg. At agiven time after the administration of the test compound, the mice wereanesthetized with diethyl ether and fluorescent light images of theentire body of the mice was photographed. For comparison, each of ICG (5mg/kg, i.v.) and the following compound (Compound A) was administeredand imaging was carried out in the same manner as above. The results areshown in FIGS. 1 to 3.

Compound 2 gave clear images of tumors at a shorter time after theadministration as compared to the reference compounds. The position oftumor was not clear within 1 hour after the administration of thereference compounds. Whilst, Compound 2 successfully gave clear imagesof the tumor at 10 to 30 minutes after the administration and revealedto be highly effective as a fluorescent contrast agent (FIG. 1).

Test Example 2 Fluorescence Imaging Test

Tumor bearing mice were prepared in the same manner as Test Example 1,and conditions for irradiation was the same as those explained in TestExample 1. Compound 5, Compound 7, and Compound 10 were used as testcompounds. Each of the test compounds (0.5 mg/ml) was dissolved inphysiological saline or phosphate buffer (pH 7.4) and administered tothe mice via a tail vein at the dose of 5.0 mg/Kg. For comparison, thefollowing compound (Compound B, 5 mg/kg, i.v.) was administered to themice.

Light was generated using a tunable, pulsed, solid state laser systemconsisting of an optical parametric oscillator (OPO) pumped by the thirdharmonic of a Nd:Yag laser (Coherent Inc.). An excitation wavelength ofλ ex=740 nm was chosen and guided with an optical fiber to the tumorbearing nude mice. The dye-specific fluorescence exitance was imagedusing a filter combination (Corion) and an intensified CCD camera (RoperScientific.) at different times after dye administration (FIG. 4).Fluorescence imagings were taken before administration, and min, 10 min30 min, 60 min, 2 hours, 4 hours, 24 hours after intravenous dyeadministration via the lateral tail venous at a standard dose of 5mg/kg. In the first 60 min, the body temperature of the animals was keptat 38° C. with heating pad. Fluorescence imaging properties of thecompounds were compared in nude mice tumor models. The results are shownin FIGS. 5 to 8. Compound 5, Compound 7, and Compound 10 gave clearimages of tumors at a shorter time after the administration as comparedto the reference compound (Compound B). The position of tumor was notclear within 1 hour after the administration of the reference compound(FIG. 8). Whilst, the compounds of the present invention successfullygave clear images of the tumor at 10 to 30 minutes after theadministration (FIGS. 5 to 7) and revealed to be highly effective as afluorescent contrast agent

INDUSTRIAL APPLICABILITY

The near infrared fluorescence contrast agent of the present inventioncan emit near infrared fluorescence by an excitation light. The nearinfrared fluorescence is superior in permeability through biologicaltissues, and therefore, the agent enables the detection of a lesion in adeep part of a living body.

1-11. (canceled)
 12. A near infrared fluorescent contrast agentcomprising a pharmaceutically acceptable injectable carrier fordiagnostic imaging and a compound of the following formula or apharmaceutically acceptable salt thereof:

wherein R¹, R², R⁷, and R⁸ independently represent a substituted orunsubstituted C₁-C₁₀ alkyl group or a substituted or unsubstituted arylgroup; or R¹ and R² and/or R⁷ and R⁸ bind to each other to form a ring;R³, R⁴, R⁵, R⁶, R⁹, R¹⁰, R¹¹ and R¹² independently represent a hydrogenatom, a substituted or unsubstituted C₁-C₆ alkyl group, a substituted orunsubstituted aryl group, a substituted or unsubstituted heteroarylgroup, a halogen atom, cyano group, carboxyl group, or sulfo group; ortwo of R³, R⁴, R⁵, R⁶, R⁹, R¹⁰, R¹¹ and R¹² bind to each other to form aring; X² represents a substituted or unsubstituted C₁-C₁₅ alkyl group ora substituted or unsubstituted aryl group; X¹ is a group represented bythe following formula

wherein X¹ and X² in total have 2 or 4 carboxyl groups; Y¹ and Y²independently represent a substituted or unsubstituted divalent linkinggroup; m¹ represents 0 or 1; m² represents or 1; m³ represents 0 or 1;L¹, L², L³, L⁴, L⁵, L⁶, and L⁷ independently represent a substituted orunsubstituted methine group, provided that when two or more of themethine groups have substituents, the substituents bind to each other toform a ring; M represents a hydrogen atom, a metal, or a quaternaryammonium salt; and n represents an integer of 1 to 7 necessary forneutralizing charge.
 13. The near infrared fluorescent contrast agentaccording to claim 12, wherein each of m¹, m², and m³ is
 1. 14. The nearinfrared fluorescent contrast agent according to claim 12, wherein X¹and X² independently represent a group represented by the followingformula:

wherein Y¹ and Y² independently represent a substituted or unsubstituteddivalent bond.
 15. The near infrared fluorescent contrast agentaccording to claim 12, wherein at least one of R³, R⁴, R⁵, R⁶, R⁹, R¹⁰,R¹¹, and R¹² is a substituted or unsubstituted aryl group or asubstituted or unsubstituted heteroaryl group.
 16. The near infraredfluorescent contrast agent according to claim 12, wherein Y₁ represents—(CH₂)_(p)CONH—, p represents an integer of 1 to 4, and Y₂ represents—(CH₂)— or (CH₂)₂—.
 17. The near infrared fluorescent contrast agentaccording to claim 12, wherein the pharmaceutically acceptableinjectable carrier for diagnostic imaging is injectable distilled water.18. The near infrared fluorescent contrast agent according to claim 12,wherein the pharmaceutically acceptable injectable carrier fordiagnostic imaging is physiological saline.
 19. The near infraredfluorescent contrast agent according to claim 12, wherein thepharmaceutically acceptable injectable carrier for diagnostic imaging isRinger's solution.
 20. The near infrared fluorescent contrast agentaccording to claim 12, wherein at least one of R³, R⁴, R⁵, R⁶, R⁹, R¹⁰,R¹¹, and R¹² is a substituted or unsubstituted aryl group.
 21. The nearinfrared fluorescent contrast agent according to claim 12, wherein atleast one of R³, R⁴, R⁵, R⁶, R⁹, R¹⁰, R¹¹, and R¹² is a substituted orunsubstituted heteroaryl group.
 22. The near infrared fluorescentcontrast agent according to claim 12, wherein Y₁ represents—(CH₂)_(p)CONH—.
 23. The near infrared fluorescent contrast agentaccording to claim 12, wherein p represents an integer of 1 to
 4. 24.The near infrared fluorescent contrast agent according to claim 12,wherein Y₂ represents —(CH₂)— or (CH₂)₂—.
 25. The near infraredfluorescent contrast agent according to claim 14, wherein each of m¹,m², and m³ is
 1. 26. The near infrared fluorescent contrast agentaccording to claim 16, wherein each of m¹, m², and m³ is 1.